Oxidase Reagent

OXIDASE REAGENT
- For in vitro use only - Catalogue No. RO95
Our Oxidase Reagent is used to detect the tetramethyl-p-phenylenediamine dihydrochloride, acts

presence of oxidase enzymes produced by a variety of as an artificial electron acceptor for the enzyme
bacteria. oxidase and is oxidized to form the colored compound
Detection of these enzymes allows for Wurster’s blue. Wurster’s blue is a purple compound
differentiation and presumptive identification of some that is readily visible and signifies a positive reaction.
bacterial species when used in conjunction with other Our formulation was devised by Kovacs and is
biochemical tests. The oxidase test can be used to often referred to as Kovacs Oxidase Reagent. The
differentiate between genera: addition of a stabilizing agent minimizes auto-
1. Moraxella (+) and Neisseria (+) from oxidation of the reagent and provides an extended
Acinetobacter (-) shelf life to the product.
2. Aeromonas (+), Plesiomonas shigelloides (+),
and Vibrio (V+) from other Enterobacteriaceae Formulation per 100 mL
(-)
Aid in species differentiation of nonenteric gram- N,N,N,N-Tetramethyl-p-phenylenediamine
negative organisms: Dihydrochloride......................................................0.60 g
1. Bacteroides distasonis (+), B. eggerthii (+), and Stabilizing Agent ....................................................0.02 g
B. ureolyticus (+) from other Bacteroides spp. (-) Dimethyl Sulfoxide (DMSO) ........................... 100.0 mL
2. Bartonella felis (+), B. quintana (w+), and B.
vinsonii (w+) from B. bacilliformis (-), B. Recommended Procedure
elizabethae (-), and B. henselae (-)
3. Bordetella parapertussis (+) from other Direct Plate Procedure
Bordetella spp. (-) 1. Allow reagent adequate time to reach room
4. Burkholderia gladioli (-) and B. mallei (V) from temperature prior to use.
B. cepacia (+) and B. pseudomallei (+) 2. Add 1-2 drops of Oxidase Reagent directly to a
5. Capnocytophaga canimorsus (w+) and C. few suspected colonies from a culture plate grown
cynodegmi (+) from C. gingivalis (-), C. on an appropriate medium such as blood agar or
ochracea (-), and C. sputigena (-) chocolate agar. (Do not flood the entire plate with
6. Haemophilus aphrophilus (-), H. parasuis (-), H. reagent)
paragallinarum (-), and H. segnis (-) from other 3. Observe for a purple color change within 30 sec.
Haemophilus spp. (+)
7. Pseudomonas syringae (-), P. viridiflava (-) from Indirect Paper Strip Procedure
other Pseudomonas spp. (+) 1. Allow reagent adequate time to reach room
8. Vibrio gazogenes (-) and V. metschnikovii (-) temperature prior to use.
from other frequently isolated Vibrio spp. (+) 2. Place a small piece of filter paper into a sterile
petri dish.
The oxidase test is based on bacterial production 3. Moisten the filter paper with 1 to 2 drops of
of an intracellular oxidase enzyme and some Oxidase Reagent.
organisms may produce more than one type of oxidase 4. Touch the colony to be tested with the end of a
enzyme. These enzymes participate in the cellular sterile wooden applicator or platinum loop.
respiration process and catalyze removal of hydrogen 5. Smear the colony onto the filter paper.
from a substrate using oxygen as a hydrogen acceptor. 6. Observe for a purple color change within 30 sec.
The active substrate in oxidase reagent, N,N,N,N-
Swab Procedure ♦ Oxidase reagent can auto-oxidize over time
therefore regular quality control checks should be
1. Allow reagent adequate time to reach room performed on known positives and negatives to
temperature prior to use. ensure adequate reagent performance
2. Saturate a piece of filter paper with Oxidase
reagent.
3. Using a sterile swab, pick a colony of interest and Quality Control
touch it lightly.
4. Rub the swab onto the filter paper. Organism Expected Results
5. Observe for a purple color change within 30 sec
on the swab, not the filter paper. Pseudomonas +ve Dark purple color
aeruginosa
ATCC 27853
Interpretation of Results Escherichia coli -ve No color change
ATCC 25922
For all procedures, a positive reaction is a purple
color change occurring within 30 seconds. Oxidase-
positive colonies typically take 10 seconds to produce Storage and Shelf Life
a positive color reaction; reactions occurring between
30 and 60 seconds should be classified as a delayed Our Oxidase Reagent should be stored at room
positive and retested. For the direct plate procedure, temperature, away from light. Under these conditions,
oxidase-positive colonies will adopt a purplish-black it has a shelf life of 16 weeks from the date of
coloration and the reagent may also impart color to the manufacture.
surrounding medium.
A negative result is no color change after 1 minute
or a color change that occurs after 1 minute. References
♦ The reagent is a clear, colorless liquid, if it 1. Gordon J, McLeod JW. Practical application of
becomes discoloured or cloudy discard the the direct oxidase reaction in bacteriology. J
reagent Pathol Bacteriol 1928; 31:185-90.
2. Kovacs N. Identification of Pseudomonas
♦ Do not attempt to perform the oxidase test on pyocyanea by the oxidase reaction. Nature 1956;
colonies grown on medium containing glucose as 178:703.
fermentation inhibits oxidase activity and may 3. Steel KJ. The oxidase reaction as a taxonomic
result in false negatives tool. J Gen Microbiol 1961; 25:297-306.
4. Isenberg HD, Ed. Clinical microbiology
♦ Do not use loops that contain iron or nichrome procedures handbook, Vol 1. Washington, DC:
since false-positive reactions may result from ASM, 1992.
surface oxidation products formed during flame 5. Koneman EW, Allen SD, Janda WM,
sterilization Schreckenberger PC, Winn WC Jr. Color atlas &
textbook of diagnostic microbiology. 5th ed.
♦ Using the oxidase test to aid in identification of Philadelphia: JB Lippincott, 1997.
Neisseria requires doing a gram stain on all 6. Forbes BA, Sahm DF, Weissfeld AS. Bailey and
oxidase-positive colonies to confirm that they are Scott’s diagnostic microbiology. 10th ed. St
diplococci Louis: Mosby, 1998.
7. MacFaddin JF. Biochemical tests for
identification of medical bacteria. 3rd ed.
Philadelphia: Lippincott Williams & Wilkins,
2000.
Original: January 2002

Revised / Reviewed: October 2014

Oxidase Reagent

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OXIDASE REAGENT

- For in vitro use only - Catalogue No. RO95

Our Oxidase Reagent is used to detect the tetramethyl-p-phenylenediamine dihydrochloride, acts

Original: January 2002

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